This invention relates to artificial DNAs which are useful in the fields of genetic engineering and medical science and to a method for DNA replication using said DNAs as the replication origin.
The replication of a linear DNA duplex unlike that of a circular DNA duplex has an intrinsic problem for completing the 5' ends of the nascent DNA strands. If a DNA polymerase uses short RNA polynucleotides as primers for the initiation of new DNA chain synthesis, removal of the RNA primer from the 5' end of the linear DNA molecule results in an irreparable gap, since all known DNA polymerases synthesize only in a 5' to 3' direction and cannot initiate de novo synthesis of a new chain. This problem was resolved by the discovery of a new DNA polymerase that uses protein as a primer.
The basic features of protein-primed initiation of DNA synthesis are now reasonably clear, and the proteins involved in the replication process have been identified in several systems, although many of the molecular details remain to be elucidated. The first step of the protein-primed DNA replication is the formation of a phosphodiester bond between the .beta.-hydroxyl group of a specific amino acid residue (serine residue in adenovirus and phage .phi.29; and tyrosine residue in bacteriophage PRD1) of a terminal protein and the first nucleotide in the new DNA chain (the complex thus formed is hereinafter referred to as the "initiation complex"). This reaction is catalyzed by the viral-encoded DNA polymerase in the presence of PRD1 DNA-terminal protein complex as a template. The nascent strand then grows by extension from the 3'-hydroxyl group of the covalently bound, first nucleotide. Successive elongation by viral DNA polymerase proceeds by concomitant displacement of the 5'-ended strand of the parental DNA. Thus, the 5' ends of the linear DNA can be preserved by the protein-priming mechanism.
Bacteriophage PRD1 is a small, lipid-containing phage, which infects a wide variety of Gram-negative bacteria. The genome of PRD1 is a linear, double-stranded DNA of about 14,700 base pairs ("bp"). A 28.times.10.sup.3 Mr terminal protein encoded by PRD1 is covalently linked to the 5' ends of viral genome. The linkage between the terminal protein and PRD1 DNA is a phosphodiester bond between a tyrosine residue of the terminal protein and dGMP, which is the terminal nucleotide for the PRD1 genome. The PRD1 DNA contains perfectly inverted terminal repeats (ITRs) of 110 to 111 base-pairs.
PRD1 DNA replication starts at either one of the DNA ends and proceeds toward the other end. The natural template for in vivo replication of PRD1 DNA is, as in the case of in vitro, the viral chromosome with the terminal protein covalently attached to the 5' end thereof (hereinafter referred to as "PRD1 DNA-terminal protein complex"). The parental terminal protein and ITR are both believed to play important roles in DNA replication as structural parts of the template. The ITRs (or part of them) seem to function as a replication origin. However, the replication mechanism of the bacteriophage PRD1 DNA has not been completely understood, and the region in ITR that acts as the replication origin has not been clarified.